Methods and compositions for modulating &#34;marginally indiscriminant&#34; hybridizations

ABSTRACT

The invention relates to the field of molecular biology, nucleic acid chemistry and medical diagnostics. More specifically, it relates to methods and compositions for promoting the hybridization of a nucleic acid probe with a target nucleic acid sequence which is not perfectly matched to the probe.

This is a continuation of U.S. application Ser. No. 09/747,164 filed onDec. 22, 2000, which is a continuation of U.S. application Ser. No.09/534,432, filed Mar. 23, 2000, which is a continuation of U.S.application Ser. No. 09/366,085, filed Aug. 3, 1999 which claimspriority from U.S. Application No. 60/095,313, filed Aug. 4, 1998.

BACKGROUND OF THE INVENTION

The ability to control hybridization of a nucleic acid strand (a probe)to its complement, while excluding imperfectly base-paired probehybridization has been central to the advancement of both molecularbiological techniques and to design of nucleic acid diagnostic systems.Much attention has been paid to this issue because identification of aparticular mutant nucleic acid sequence fully complementary to a probecan permit detection of, for example, the existence of a mutant sequence(genetic disorders) or a particular virulent bacterial or viral strainin a patient. Thus, is studies of the physics of mismatched probe:targetenergetics has focused on the difference in free energy of suchmismatches with the hope that such knowledge will benefit thedevelopment of assays in which such mismatches are excluded. Forexample, in diagnosis of a genetic disorder a mutant probe for targetinga nucleic acid molecule having a sequence containing a single basemismatch associated with such a disorder would produce a false positiveresult if the probe also hybridizes to the wild-type (unmutated)sequence. In other words, the assay must be sufficiently discriminatoryin order for the probe to bind to the molecule having the mutated baseand not to the molecule lacking the mutated base. If the probehybridizes to both molecules, then the hybridization result wouldindicate the presence of the single base mutated sequence even though itwas not, in fact, contained in the tested sample. In general, theenvironment of the reaction is manipulated to eliminate such mismatchprobe:target interactions by modifying the physical conditions forhybridization (e.g., temperature and or time) or composition of thehybridization buffer (e.g., salt, divalent ions denaturing agents,etc.).

On the other hand, however, it is advantageous, in some applications, tohave a probe which is known to hybridize with molecules containingparticular mismatched sequences (a “marginally indiscrimninant” probe)within a desired degree of homology to the probes' perfect complement.This would permit a single probe to be used in an assay for determiningthe presence of nucleic acid molecules containing any of the mismatchedsequences. Such an assay would thus reduce, or possibly even eliminate,the need for more than one probe, each containing a nucleic sequenceprecisely corresponding to a sequence of a target molecule. Achievementof such a probe could be useful as a “multiplex” (multiple assays fromone probe) probe. To date, for example, conventional multiplexing hasrelied upon the inclusion of multiplex specific probes into one cocktailreaction (e.g., multiplex polymerase chain reaction (PCR)), rather thanjust one probe.

There are always going to be constraints on an indiscrimninant probe. Itwould be generally acceptable for a probe to hybridize to any nucleicacid molecule whether complementary or not (although there may belimited use for such a probe in detecting the presence or absence of anyDNA). This type of probe and/or conditions for hybridization of theprobe would detect even sequences which shared no homology with theprobes' complement. At the other end of the spectrum, it is desirablethat when designing a marginally indiscrimninant probe for detectingviral nucleic acid sequences, for example, to design a probe such that asingle probe will pick up all known of sequence within a limited degreeof homology (say 10, 20, 30, 40 or 50% homology).

There are known approaches for detecting target nucleic acids byhybridization of a probe having a nucleic acid sequence fullycomplementary to or substantially complementary to a sequence of atarget nucleic add. Methods have thus been developed to detect viralnucleic acid sequences and their variants by hybridization using probesfully complementary to or substantially complementary to the viralnucleic acid sequences, as exemplified by U.S. Pat. Nos. 5,008,182;5,079,351; 5,268,268; 5,567,603; 5,594,122; 5,594,123; 5,599,662; and5,733,781, the text of which is incorporated herein by reference.

The specifications of these patents disclose methods and compositions ofnucleic acids for as probes for detecting nucleic acid sequences of thefamily of Human T-cell Leukemia Viruses (HTLV) and the HumanImmunodeficiency Virus (HIV). HIV and its variants are thought to beresponsible for the acquired immunodeficiency syndrome (AIDS). Theprobes and methods disclosed in these patents for detecting the presenceor absence of the viral DNA utilize probes to conserved regions of theseviruses, but the disclosed approaches have limited applicability. Thisis because of the now well-known genetic variability of humanimmunodeficiency viruses. Genetic variations arise with high frequency.This variability has complicated the development of assays for detectingthe presence of their genetic material. Further, while a comparison ofvarious HIV-1 isolates has revealed, regions of the genome that arereasonably well conserved, it is possible that even the conservedregions, regions to which the probes have been designed to hybridize,may at mutate in the future. If so, probes designed for detecting theconserved regions may not hybridize to the one is conserved region as aresult of base mismatches.

As a further example, U.S. Pat. No. 5,567,603 describes probes fordetecting HIV-3 that hybridize neither with the sequences of HIV-1 norwith the sequences of HIV-2 under stringent hybridization conditions.Thus, the ability to design a single nucleic acid probe and a methodthat will allow hybridization of the probe to all HIV strains and theirvariants but not to other non-target partially complementary nucleicacid sequences or other non-related viral nucleic acid sequences wouldhave advantages over current approaches.

SUMMARY OF THE INVENTION

The present invention describes how a nucleic acid probe with mismatchesto a target may be forced to hybridize to a target without hybridizingindiscriminantly with other non-target partially complementary nucleicacids. The methods of the invention require that nucleic acid duplexligands as well as nucleic acid single-strand ligands be titered inconcentration against one another to achieve the required degree ofmismatch target hybridization without obtaining non-targethybridization.

In one aspect of the invention, a method of providing a nucleic acidmolecule for potential use as a probe for a family of nucleic acidmolecules in the present of a nucleic acid sequence binding ligand whichwill promote hybridization of the probe to all the member of the familyof target sequences and not to non-target partially complementarysequences is provided. The method includes the steps of providing thefamily of first nucleic acid molecules wherein each member of the familyis related to all other members of the family by a consensus sequence. Asecond nucleic acid molecule complementary to the consensus sequence issynthesized by methods well known in the art. It is highly preferablethat the homology of this complementary sequence to other viral nucleicacid sequences as well as other sequences in general be determined bycomparing its nucleotide sequence against those listed in a database(e.g., GenBank, DDBJ, EBI, or GSDB) to ensure that it does not by chancehappen to have significant homology to other non-target partiallycomplementary sequences. In addition, the homology of The complementarysequence should also be searched against all members of the family oftarget sequences to determine if the probe might hybridize to aregion(s) other than it was originally intended to. Once it isdetermined that the complementary sequence will most likely nothybridize to a region(s) of the target sequence and all its familymembers other than it was intended to or to other non-target partiallycomplementary nucleic acid sequences, the nucleic acid sequencecomplementary to the consensus sequence can be used as a probe. Theability of the probe to hybridize to the consensus region of the targetnucleic acid sequence and all members of the family is then determinedin the presence of a certain concentration of nucleic acid sequencebinding ligand known to affect hybridization of the probe to thecomplementary region of the target sequence. This is repeated at severaldifferent concentrations of ligand, such that the concentration ofligand at which the probe is able to bind to the target nucleic acidsequence and all its family members equally well without affecting thehybridization of the probe to other non-target partially complementarynucleic acid sequences is the concentration of ligand that will be usedfor subsequent methods of the invention for that particular probe indetecting the presence of the its target nucleic acid sequence and itsgenetic variants.

In yet another aspect, hybridization of the probe to the target nucleicacid sequence and its family members can be further improved in thepresence of two different nucleic acid binding ligands.

In a preferred embodiment, a method of promoting the hybridization of anucleic acid capture moiety comprising a nucleic acid sequencecomplementary to a consensus sequence of a target single-strandednucleic acid sequence and all its family members without hybridizing toa plurality of other non-target partially complementary nucleic acidsequence suspected of being present in a sample is provided. The methodincludes the steps of identifying at least one consensus sequence to aregion of the target duplex nucleic acid sequences and all its geneticvariants suspected of being present in a sample; synthesizing a nucleicacid sequence complementary to the consensus sequence (probe); providinga nucleic acid capture moiety comprising the probe; a nucleic acidbinding ligand, wherein the ligand has been selected to promotehybridization of the probe to the corresponding complementary region ofthe target nucleic acid and all its family members; the sample suspectedof containing the target nucleic acid sequence and all its familymembers; and allowing the target single-stranded nucleic acid sequenceand all its family members to the nucleic acid capture moiety comprisingthe probe without promoting the hybridization of other non-targetpartially complementary nucleic acid sequences.

In yet another preferred embodiment, a consensus sequences of the targetsequence and all its family members can be amplified by PCR if it issuspected that direct detection of the target nucleic acid sequences andall its family members may be different or impossible. In this case, thenucleic acid primers used for detecting the target nucleic acidsequences and all its family members should be designed such that theprimers will allow amplification of the region of target nucleic acidsequence and all its family members (i.e., the consensus sequence) to bedetected without simultaneous amplification of non-target partiallycomplementary nucleic acid sequences from other viral nucleic acidsequences or human genomic nucleic acid sequences. The hybridization ofthe amplified region of the target sequence and all its family membersto a nucleic acid capture moiety comprising the probe can then beperformed under conditions in which the presence of a nucleic acidsequence binding ligand will promote hybridization of the targetsingle-stranded nucleic acid sequence and all its family members to thenucleic acid capture moiety without promoting the hybridization of othernon-target partially complementary nucleic acid sequences.

In a preferred embodiment, the invention described herein can be used todetect nucleic acid sequences of the AIDS virus and all of its familymembers without detecting other non-target viral nucleic acid sequencesor human genomic DNA.

In yet another preferred embodiment, the invention described herein canbe used to detect nucleic acid sequences associated with infectiousdiseases, genetic disorders, or cellular conditions such as cancer inwhich the gene responsible for the pathological condition is known to becaused by several mismatch variant nucleic acid sequences. Examples ofsuch genes include but are not limited to p53, ras, BRCA1, BRCA2, orAPC.

In another embodiment, the invention herein relates to a multi-containerkit for detecting target nucleic acid sequences and their mismatchnucleic acid sequences suspected of being present in a sample, which kitcomprises:

-   -   (a) a nucleic acid capture moiety comprising a labeled probe        nucleic acid sequence substantially complementary to a consensus        sequence of the target duplex nucleic acid sequence and all its        family members variants suspected of being present in a sample;        and    -   (b) at least one nucleic acid sequence binding ligand, wherein        the ligand promotes hybridization of the target single-stranded        nucleic acid sequence and all its family members to the nucleic        acid capture moiety comprising the labeled probe sequence        without promoting the hybridization of other non-target        partially complementary nucleic acid sequences.

Preferably, the kit also contains nucleic acid sequences primers foramplifying the region of the target nucleic acid sequence and all itsfamily members to be detected, an agent for polymerization and fourdifferent nucleosides. It is also preferable that the kit contain therelevant positive and/or negative controls.

In preferred embodiments, the label of the labeled probe nucleic acidsequence is then selected from the group consisting of antibody,antigens, radioisotopes, fluorescent, enzyme, lecithin or biotin.

In a preferred embodiment, the components of the kit are designed todetect the AIDS virus and all its family members without detectingnon-target viral nucleic acid sequences and other non-target nucleicacid sequences.

In another preferred embodiment, the components of the kit are designedto detect nucleic acid sequences associated with infectious diseases,genetic disorders, or cellular conditions such as cancer in which thegene responsible for the pathological condition is known to be caused byseveral mismatch variant nucleic acid sequences.

Examples of such genes include but are not limited to p53, ras, BRCA1,BRCA2, or APC.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect of titrating of four DNA binding ligands in aDNA hybridization reaction in which the target molecules were labeledwith the radioisotope ³²P. Dark bands indicate unbound target (i.e.,higher band intensity=less bound target). The control lane shows thetotal intensity of unbound target. Hybridization was allowed to occurunder the conditions described herein. At the end of the specified timeperiod for hybridization, the amount of unbound target was determined bygel electrophoresis.

FIG. 2 shows the effect of titrating combinations of four DNA bindingligands in a DNA hybridization reaction in which the target moleculeswere labeled with ³²P. Distamycin A was held constant at 1 mM for thosesets with drug combinations. Hybridization was allowed to occur underthe conditions described herein. At the end of the specified time periodfor hybridization, the amount of unbound target was determined by gelelectrophoresis.

FIG. 3 a shows the time dependence of target-probe hybridization in thepresence and absence of distamycin A and ethidium bromide. The arrowindicates the perfect matched probe. The hairpin and target sequencesare listed in the inset. At the end of the specified time period amountof unbound target was determined by gel electrophoresis.

FIG. 3 b shows graphs of normalized binding curves from the dataobtained in FIG. 3 a. Gray circles indicate hybridization without DNAligands. Black circles indicate hybridization in 1 mM distamycin and0.001 mM ethidium bromide.

FIG. 4 shows the effect of salt concentration dependence of denaturationof target:probe hybrids of 40% (v/v) formamide. The buffer was 10 mMphosphate, pH 7.2 and the specified concentration of NaCl. Washincubation time was held constant at 1 hr. The amount of unbound targetat the end of the wash incubation time was determined by gelelectrophoresis.

FIG. 5 a shows the effect of formamide concentration when cross-titteredwith distamycin A in the wash buffer on denaturation of target probehybrids. Darker bands indicate a higher degree of dissociation. At theend of the wash period, the amount of ³²P labeled target was determinedby gel electrophoresis.

FIG. 5 b shows graphs of binding curves from the data obtained in FIG. 5a with each of the constructs shown.

FIG. 5 c shows graphs of the fraction of target:probe hybrids remainingafter denaturation as a function of formamide at differentconcentrations of distamycin A with each of the constructs shown.

DETAILED DESCRIPTION OF THE INVENTION

The methods and unique compositions of the invention are useful in thedetection of nucleic acid sequences and all related family memberswithout detecting non-target partially complementary nucleic acidsequences. The methods of the invention can be performed with nucleicacid capture moieties immobilized on a solid support such as multi-wellplates, membranes or gene chips. The methods of the invention can alsobe automated, in part, to speed screening and improve economy.

The term “single-stranded nucleic acid”, as used herein, refers to aduplex nucleic acid which has been denatured resulting in twosingle-stranded nucleic acid sequences of DNA or RNA. Methods ofdenaturing duplex nucleic acid sequences are well known to those skilledin art. Single-stranded nucleic acid can also mean a mixed DNA-RNAstrand, or nucleic acid-like compounds such as peptide nucleic acids. Anucleic acid strand can also include modified (e.g., chemically orbiochemically modified) DNA or RNA bases, of which many are known in theart.

The terms “target nucleic acid sequence”, “target nucleic acid” or“target strand” refer to a nucleic acid sequence which is to bedetected, sequenced, immobilized, or manipulated. The target nucleicacid sequence can be any nucleic add strand, as defined above, and ingeneral will be single-stranded or will be made single-stranded bymethods known to those skilled in the art. The target nucleic acidsequence can be obtained from various sources including plasmids,viruses, bacteria, fungi, yeast, plants, and animals, including humansor the target nucleic acid sequence can be obtained from non-naturalsources. The target nucleic acid sequence can be obtained from variousorganisms or tissues, including fluids such as blood, semen, urine andthe like. The target nucleic acid sequence is preferably extracted orpurified to remove or reduce contaminating or interfering materials suchas proteins or cellular debris. Procedures for such purification orextraction of target nucleic acids sequences are known in the art,including, for example, those described in Maniatis et al., “MolecularClorung: A Laboratory Manual”, Cold Spring, Harbor Laboratory (1989), orin Bell et al., Proc. Nat Acad. Sci. USA (1991), 78:5759-576. Themethods and compositions of the inversion are particularly useful in thedetection of nucleic acid sequences associated with infectious diseases,genetic disorders, or cellular conditions such as cancer.

In one aspect, the invention features a nucleic acid capture moietywhich has at least one nucleic acid sequence complementary to at leastone consensus sequence of a target nucleic acid sequence and having atleast two nucleic acid sequence regions which are capable of forming anintramolecular duplex. The capture moiety can be immobilized on thesolid support before, simultaneous with, or after capturing thesingle-stranded target nucleic acid sequence. A nucleic acid capturemoiety can “capture” a target nucleic acid sequence by hybridizing tothe target nucleic acid sequence and thereby immobilizing the targetnucleic acid sequence and all its family members. In preferredembodiments, the nucleic acid capture moiety comprises a nucleic acidsequence strand which has at least one nucleic acid sequence which iscomplementary to a consensus sequence of the target nucleic acid and allits family member. One example of a nucleic acid capture moiety is anucleic acid hairpin. A “hairpin” is a double-helical region in a singleDNA or RNA strand formed by the hydrogen bonding between adjacentinverse complementary sequences along the nucleic acid strand. The useof a nucleic acid hairpin as a nucleic acid capture moiety has beendescribed in detail in U.S. Pat. No. 5,770,365 issued to the currentapplicants, the disclosure of which is incorporated herein by reference.In certain embodiments, the nucleic acid sequence capture moiety,whether a single-stranded nucleic acid sequence or a nucleic acidhairpin, may be labeled as with, e.g., a radioisotope, a fluorescentmoiety, an antibody, an antigen, a lecithin, an enzyme, biotin or otherlabels well known in the art. Alternatively, the target sequence mayalso be labeled or labeled secondary probes may be employed. A“secondary probe” is a nucleic acid sequence which is fullycomplementary or substantially complementary to a region of the targetnucleic acid sequence or to a region of the nucleic acid capture moiety.“Substantially complementary” as used herein means that the sequencemust be sufficiently complementary to the nucleic acid being detectedsuch that hybridization will take place under the conditions employed.Alternatively, a nucleic acid capture moiety can also be a linearnucleic acid sequence such as a single stranded DNA or RNA nucleic acidcomprising at least one nucleic add sequence which sequence iscomplementary to the consensus sequence to the consensus sequence to allmembers of the family of target sequences.

As used herein, the term “consensus sequence” means an idealizedsequence that represents the nucleotides most often present at eachposition in a given segment of all members of the family of targetsequences. One method of determining a consensus sequence is to use acomputer program to compare the target nucleic acid sequence and all itsfamily member sequences for which a consensus sequence is desired. Forthis purpose, a commercial program with the underlying computeralgorithm provided by the National Biomedical Research Foundation usinga dot matrix may be conveniently employed. The program involvesinputting the nucleic acid sequences of the target nucleic acid sequenceand all its generic variants and defining a window size for base pairhomology. The program employs graphics to compare the sequences ondifferent axes, and a dot appears where there is at least substantialhomology. As used herein, the term “target nucleic acid sequence and allits genetic variants” refers to the wild-type nucleic acid sequence andall base mismatch variants of the wild-type sequence. Once the consensussequence has been determined a nucleic acid sequence complementary tothe consensus sequence is synthesized by methods well known in the art.It is preferable, however, that prior to synthesizing the complementarysequences, the complementary sequence be searched against a plurality ofnucleic acid sequences listed in one or more of the nucleic acidsequence databases which include but is not limited to the DNA Data Bankof Japan (DDBJ), the European Bioinformatics Institute (EB), GenBank, orthe Genome Sequence Database (GSDB) to determine if the complementarysequence has significant homology to other non-target partiallycomplementary nucleic acid sequences. If the consensus sequence happensby chance to have significant homology to another non-related viralnucleic acid sequence or to an unrelated human sequence, a new consensussequence to another region of all members of the family of targetsequences can be selected and the above process repeated. By doing this,false positives can be eliminated. It is also preferable that thecomplementary sequence be searched against all members of the family oftarget sequences to determine if the complementary sequence mighthybridize to a region(s) other than it was originally intended to. Onceit is determined that the complementary sequence will most likely nothybridize to a to a region(s) of the target sequence and all its familymembers other than it was intended to or to other non-target partiallycomplementary nucleic acid sequences, the nucleic acid sequencecomplementary to the consensus sequence can be synthesized and used as aprobe. Otherwise, a new consensus sequence should be selected and theabove process repeated.

“Sequence identity or homology”, as used herein, refers to the sequencessimilarity between two nucleic acid molecules. When a position in bothof the two compared sequences is occupied by the same base, e.g., if aposition in each of two DNA molecules is compared by adenine, then themolecules are homologous or sequence identical at that position. Thepercent of homology or sequence identity between two sequences is afunction of the number of matching or homologous identical positionsshared by the two sequences divided by the number of positionscompared×100. For example, if 6 of 10, of the positions in two sequencesare the same, then the two sequences are 60% homologous or have 60%sequence identity. By way of example, the DNA sequences ATTGCC andTATGGC share 50% homology or sequence identity. Generally, a comparisonis made when two sequences are aligned to give maximum homology. Unlessotherwise specified “loop out regions”, e.g., those arising fromdeletions or insertions in one of the sequences are counted asmismatches.

The comparison of sequences and determination of percent homologybetween is two sequences can be accomplished using a mathematicalalgorithm. Preferably, the alignment can be performed using the ClustalMethod. Multiple alignment parameters include GAP Penalty=10, Gap LengthPenalty=10. For DNA alignments, the pairwise alignment parameters can beHtupla=2, Gap penalty=5, Window=4 and Diagonal saved=4. For proteinalignments, the pairwise alignment parameters can be Ktuple=1, Gappenalty=3, Window=5, and Diagnosis Saved=5.

Additional non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the algorithm of Karlin and Altachu(1990) Proc. Natn. Acad. Sci. USA 87:2264-68, modified as in Karlin andAltachu (1993) Proc. Natn. Acad. Sci. USA 90:5873-77. Such an algorithmis incorporated into the NBLAST and XBLAST programs (version 2.0) ofAltachu, et al., (1990) J. Mol Biol. 215:403-10. BLAST nucleotidesearches can be performed with the NBLAST program, score=100wordlength=12 to obtain nucleotide sequences homologous to nucleic acidmolecules of the invention. BLAST protein searches can be performed withthe XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to protein molecules of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altachu et al., (1977) Nucleic Acids Research25(17):3389-3402. When utilizing BLAST and Gapped GLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be sued. See http://www.nebi.nlm.nih.gov. Another preferrednon-limiting example of a mathematical algorithm utilized for thecomparison of sequences is the algorithm of Myers and Miller, CABIOS(1989). Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used.

The compositions and methods of the invention generally feature the useof at least one base-preferring binding ligand (or, in some cases,sequence-specific ligand) to promote hybridization of a probe nucleicacid sequence to promote hybridization of the target single-strandednucleic acid sequence and all its family members to the is nucleic acidcapture moiety without promoting the hybridization of other non-targetpartially complementary nucleic acid sequences. The methods andcompositions of the invention can also include one or more additionalbinding ligands, which can be base-preferring or sequence-specificligands, or non-specific ligands, and can bind duplex nucleic acidsequences or single-stranded nucleic acid sequences. The term“nonspecific binding ligand”, as used herein, refers to a nucleic acidbinding ligand that does not substantially preferentially bind tonucleic acid sequences in which one or more specified bases predominate.That is, a “nonspecific binding ligand” binds to all, or a large varietyof, bases or sequences approximately equally well. The choice ofappropriate ligands will be routine to the skilled artisan in light ofthe teachings herein, as explained in more detail below.

Ligands suitable for use in the present invention are capable, ingeneral, of binding to nucleic add single strands and/or duplexes. Ingeneral, it is necessary to provide at least one base-preferring ligandin the reaction mixtures of the invention.

A variety of base-preferring ligands have been described. For example,the duplex-binding ligand distamycin A has been reported to bindpreferentially to AT-rich sequences. Other base-preferring,duplex-binding ligands include certain restriction enzymes, drugs suchas actinomycin D (which has a primary binding site of 5′-GC-3′, and asecondary preference for GT sites) and intercalators such as ethidiumbromide (as described below).

Similarly, base-preferring single strand-binding ligands can be employedin the invention.

The method of the invention is particularly useful for detecting geneticvariants of a target nucleic acid sequence by hybridization using asingle probe in the presence of a pre-selected nucleic acid bindingligand under conditions such that the nucleic acid binding ligand willpromote hybridization of the target nucleic acid and all its geneticvariants with the probe but not to other non-target partiallycomplementary nucleic acid sequences. Specifically, the method of theinvention can be used to detect the presence of the AIDS virus nucleicacid sequence and all its genetic variants. A candidate consensussequence to a particular region of the viral nucleic acid sequence andall its family members is first selected. A second nucleic acid sequencecomplementary to the consensus sequence, the probe, can then besynthesized by methods well known in the art. It is preferable that thenucleic acid sequence of the probe be compared to a plurality of nucleicacid sequences in a database to rule out the possibility that othernon-related viral nucleic acid sequences or other nucleic acid sequenceswith significant homology may hybridize to the probe, resulting in falsepositives. The conditions under which a nucleic acid ligand will promotehybridization of the probe sequence to the AIDS virus nucleic acidsequence and all its family members is then determined. A second nucleicacid ligand different from the first can also be used to further improvehybridization of the probe to the AIDS virus nucleic acid sequence andall its genetic variants without promoting hybridization of the probe toother non-target partially complementary sequences. If it is suspectedthat the amount of AIDS viral nucleic acid sequence present is below thelevel of direct detection of the method herein, the consensus sequencevan be amplified by PCR using consensus sequence primers immediatelyadjacent to the region to be detected.

Similarly, the method of the invention can be used to detect thepresence of any nucleic acid sequences associated with infectiousdiseases, genetic disorders, or cellular conditions such as cancer inwhich the gene responsible for the pathological condition is known to becaused by several mismatch variant nucleic acid sequences. Examples ofsuch genes include but are not limited to p⁵3, ras, breast cancerantigen 1 (BRCA1), or breast cancer antigen (BRCA2).

The present invention will now be illustrated, but is not intended to belimited by the following examples:

General Methods

A. Constructs Used

The biotinylated DNA capture hairpin (hairpin) was purchased from acommercial supplier (Oligos Therapeutics), wit the following structure: TTCCTGGTGCAGCTGATC-5′ / U* \  TTGGACCACGTCGACTAGGGCTCCTCTGCGATCCATA-3′

The duplex region will henceforth be referred to as the “stem.” The 5bases forming a single-stranded loop on one end of the hairpin will bereferred to as the “hairpin loop” or “loop.” “U” refers to biotinylatedU, used for attaching the hairpin to a solid support (in this case,streptavidin-coated microtiter plates). The single-stranded region(shown above in bold-face) will be referred to as the “dangling end.”

Single-stranded DNA molecules fully or partially complementary to thedangling end (referred to as “probe”) were also purchased from the samesupplier. These molecules were of different lengths to allow them to beseparated and visualized by PAGE. The sequences are: 15-mer perfectmatch: 5′-TAT GGA TCG GCA GAG-3′ 17-mer mismatch: 5′-AT TAT GGA TCG GCAGAT-3′ 19-mer mismatch: 5′-AAAT TAT GGA TCG GCG GAG-3′ 21-mer mismatch:5′-TAAAAT TAT GGA TCT GCA GAG-3′ 23-mer mismatch: 5′-TTTAAAAT TAT GGGTCG GCA GAG-3′

Note that the mismatches are longer than the 15-mer perfect-matchedsequence on the 5′-end.

The duplex molecules formed are: 1. TTCTGGTGCAGCTGATC-5′ GAGACGGCTAGGTAT-5′ / U* \ TTGGACCACGTCGACTAGGGCTCCTCTGCGACCATA-3′ 2. TTCTGGTGCAGCTGATC-5′ TAGACGGCTAGGTATTA-5′ / U* \ TTGGACCACGTCGACTAGGGCTCCTCTGCGATCCATA-3′ 3. TTCTGGTGCAGCTGATC-5′ GAGGCGGCTAGGTATTAAA-5′ / U* \ TTGGACCACGTCGACTAGGGCTCCTCTGCGATCCATA-3′B. Reaction Conditions1. ³²P Labeling of the Target Molecules

The five target molecules were labeled with ³²P following a standardkinasing protocol. The labeled bands were isolated from the reactionsolutions by denaturing PAGE (8 M. urea, 20% actylamide). ³²P activitywas determined by scintillation counting.

2. Capture Hairpin Immobilization on Microtiter Plates

A solution of the capture hairpin at 10 pmol/50□1 in PBS (150 mM NaCl,10 mM phosphate, pH 7.2) was prepared. 50□1/well was loaded onstreptavidin-coated microtiter plates (Boehringer-Mannheim #1645692) andallowed to incubate for 30 min at room temperature. After the incubationperiod, the wells were washed 6 times with PBS, and blotted on cleanKimwipes.

3. General Procedure for Hybridization

A cocktail of the labeled targets was prepared by adding a sufficientamount of each target to the hybridization mixture to give a finalconcentration of −20,000 cpm/target/25□1. The final composition ofhybridization mixture is 1M NaCl, 10 mM phosphate, pH 7.2, and thespecified concentration of the ligand. 25□1 of the target cocktail wasloaded into each well and the plate was incubated for the specifiedamount of time. After incubation, each reaction mixture asquantitatively transferred to a 0.2 ml tube (Costar 6547).

The samples were analyzed by denaturing PAGE as follows: 10□1 of loadingdye (8 M urea, 5 mM Tris-HC1, pH 7.5, 100 mM EDTA, 0.01% bromophenolblue, 0.01% xylene cynol) was added to the tube, and the whole samplewas loaded onto a 15% acrylarnide/1×TBE/7 M urea gel. PAGE was run at 20mA/gel for 2 hours. After electrophoresis, the gels were visualized byautoradiography.

4. General procedure for denaturation

The hybridization mixture was incubated typically for 2 hours, under thespecified conditions (i.e., hybridization buffer+ligands). Afterincubation, the reaction mixture was removed, and the wells washed oncewith 100 μl 1M NaC1 phosphate, pH 7.2. The plate was blotted onKimwipes, and 50 μl of the specified denaturation buffer was added andallowed to incubate for the specified amount of time. The mixture wasthen quantitatively transferred to 0.2 μl of loading dye was added, andthe sample analyzed by PAGE as above.

The autoradiograms were done by exposing X-ray films (Kodak X-OMAT) tothe gels overnight, using an image intensifying screen. In some cases,there is a lane marked “control.” This is a reference lane loaded withan equal volume (25 μl) of unhybridized target cocktail. Also, in eachdenaturation set of experiments, there is a lane marked “initial.” Thislane was loaded with the reaction mixture after hybridization, whichindicates how much of the target has bound.

EXAMPLE 1 Effect of Single Ligands of Hybridization

An hybridization experiment was done where the following binders (seeTable 1) were titrated: actinomycin D. distamycin A, ethidium bromide,and single-strand DNA binding protein (SSB). Incubation time was heldconstant at 2 hours. The results are shown in FIG. 1.

Results

-   1. Addition of actinomycin D to the hybridization reaction decreased    the extent of hybridization in all cases. It acted as a    single-strand binder (i.e., denaturant), with the activity    proportional to the concentration.-   2. Distamycin A improved binding up to a concentration of 0.016 mM,    but did not improve binding (compared to the control with no    ligands) at higher concentrations.-   3. Ethidium bromide died not seem to affect the extent hybridization    up to a concentration of 0.001 mM, and it inhibited hybridization of    the longer mismatches (19-21, 23-mer sequences) from a concentration    of 0.004 mM and higher. There are no bands at 1 mM. However, there    was a strong band at the top of the gel (data not shown).-   4. SSB did not have an effect on the hybridization up to a    concentration of 0.78 μg/ well. However, a decrease in the extent    hybridization was observed at the higher SSB concentrations.

EXAMPLE 2 Effect of a Ligand Combination of Hybridization

In this experiment, different combination of ligands were used. Thetitration of distamycin A was repeated (see above), and in three othersets, distamycin A concentration was fixed at 1 mM and the other ligandswere titrated. The results are sown in FIG. 2.

Results

-   1. The distamycin A titration experiment showed nearly identical    results with the first run. An improvement in the extent of    hybridization was observed up to a concentration of 0.016 mM, with    no improvement at higher concentrations.-   2. Titration of actinomycin D in the hybridization mix with a    constant amount of distamycin A showed markedly different results    than when actinomycin D was used alone. A comparison of the two    experiments (II-1 and III-2) showed that when actinomycin D was used    alone, a decrease in the extent of hybridization was apparent even    at the lowest concentration used (0.00025 mM). When actinomycin D    was used in combination with distamycin A, a decrease in the extent    of hybridization was noted at 0.004 mM or higher, an approximately    16-fold higher concentration.-   3. A combination of distamycin A and ethidium bromide showed a    similar effect While there was a decrease in the hybridization    at >0.001 mM ethidium bromide when it was used alone, there was no    decrease in hybridization when it was used in combination with    distamycin A. Similar to the previous experiment, at 1 mM ethidium    bromide, all the unhybridized target was noted at the top of the gel    (data not shown).-   4. Distamycin A apparently did not have an effect on the activity of    SSB. The results of the distamycin A/SSB combination are similar to    the results when SSB alone was titrated.

EXAMPLE 3 Effect of a Ligand Combination on Hybridization Time

The previous experiment showed that a combination of ligands (i.e.,distamycin A and ethidium bromide) may improve the extent of DNAhybridization. A hybridization kinetics experiment was performed wherethe extent of hybridization in the absence of ligand (i.e.,hybridization buffer only) and with a combination of ligands (1 mMdistamycin A+1 μM ethidium bromide) were compared as a function of time.

Results

The results are shown in FIG. 3 a. A comparison of the band intensitiesat 40 and 60 minutes shows an improvement in the hybridization in thepresence of ligand. This trend is more clear when the intensities aremeasured (NIH Image) and plotted as shown in FIG. 3 b.

EXAMPLE 4 Effect of Denaturation on Hybridization

In this set of experiments, we used various combinations of saltconcentration, distamycin A, and the formamide (a denaturant), tocontrol the extent of duplex to single-strand dissociation. The same setof molecules as in the previous section was used.

Hybridization of the target cocktail to the capture hairpin was carriedout following the procedure described in General Methods. The finalcomposition of the hybridization buffer was 1M NaC1, 10 mM phosphate, pH7.2. The samples were incubated for approximately 2 hours at roomtemperature, and washed once with the hybridization buffer.

The denaturation buffer was 10 mM NaCl, 10 mM phosphate, pH 7.2, andNaCl tittered from 0 to 1M. The results are shown in FIG. 4. The amountof target dissociating from the capture hairpin decreased with anincrease in the salt concentration with the 15-mer perfect match showingthe greatest change.

Results

-   1. Salt Concentration Dependence of Denaturation

The wash buffer consisted of 40% formamide, 10 mM phosphate pH 7.2, andNaC1 tittered from 0 to 1M. The results are shown in FIG. 4. The amountof target dissociating from the capture hairpin decreased with anincrease in the salt concentration, with the 15-mer perfect matchshowing the greatest change.

-   2. Formamide and Distamycin A Concentration Effect on Dissociation

FIG. 5 a shows a denaturation experiment where formamide wascross-tittered with distamycin A. The buffer concentration was keptconstant at 10 mM NaCl, 10 mM phosphate pH 7.2 Formamide was titteredfrom 20-35% at 2.5 increments, while distamycin A was tittered from0.062 mM to 1 mM in 4-fold increments. With no distamycin A, thestabilities of the mismatched targets increased, as shown by thedecrease in their respective band intensities. This effect becomes moreclear when the bands are quantified and plotted, as shown in FIG. 5 b(with distamycin A independent variable), and in FIG. 5 c (withformamide as the dependent variable).

-   3. Time Dependence of Dissociation as a Function of Distamycin A    Concentration

An experiment was fun where the extent of dissociation over time wasmeasured as a function of distamycin A concentration in the wash buffer.The formamide concentration was kept constant at 40% (v/v), anddistamycin A was tittered in 4-fold increments, at 0,0,62 mM, 0.25 mM,and 1 mM. Time points were from 0 (wash buffer was added and pulled out)to 60 min. The results are shown in FIG. 6. With 0-0.062 mM distamycinA, denaturation dissociate to the same extent as in the previous drugconcentration. At 1 mM distamycin A, all target molecules show a lowerextent of dissociation, with the perfect match showing a marked increasein stability.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of this invention and are covered by the followingclaims. The contents of all references, issued patents, and publishedpatent applications cited throughout this application are herebyincorporated by reference.

1. A method of identifying a nucleic acid molecule suitable for use in aprobe for detecting the presence of one or more of a family of nucleicacid molecules, comprising the steps of: (a) providing the family offirst nucleic acid molecules wherein each member of the family isrelated to all other members of the family by consensus sequence; (b)providing a second nucleic acid molecule having a sequence complementaryto the consensus sequence; (c) determining the ability of the secondnucleic acid molecule to form a duplex with each member of the family inthe presence of a first ligand known to affect duplex formation ofnucleic acid molecules; (d) repeating step (c) for a plurality ofconcentrations of the ligand, wherein the nucleic acid molecule suitablefor use in a probe is identified in step (c) at a ligand concentrationat which the ability of the second nucleic acid molecule to form aduplex with each member of the family is substantially the same as itsability to form a duplex with each other member of the family.
 2. Themethod of claim 1, further comprising the step of (e) repeating steps(c) and (d) for a second ligand.
 3. The method of claim 2, furthercomprising the step of (f) repeating steps (c) and (d) in the presenceof both first and second ligands.
 4. The method of claim 3 wherein step(e) is repeated for each of a plurality of the second ligands and step(f) is repeated for each of the second ligands or combinations thereof.5. The method of claim 1, further comprising the step of (g) determiningthe percent homology of the second nucleic acid sequence against aplurality of nucleic acid sequences in a database prior to step (c),wherein the second nucleic acid sequence is less than a predeterminedhomology to other non-target partially complementary nucleic acidsequences.
 6. The method of claim 1, wherein the first and secondligands are selected from the group consisting of actinomycin D,distamycin A, diminazane aceturate, bisbenzimide, and ethidium bromide.7. The method of claim 1, wherein said first nucleic acid molecules ofthe family are at least a % homologous with each other, a being a numbergreater than 0 and less than 100, comprising the further steps of: (i)providing a third nucleic acid molecule which is no more than b %homologous with each of the first nucleic acid molecules of the family,where b is a number greater than 0 and less than a; (ii) repeating steps(c) and (d) in the presence of the third nucleic acid molecule so as to:A. determine the ability of the second nucleic molecule and is the thirdnucleic molecule to form a duplex with each other, and B. determine aligand concentration at which the ability of the second nucleic acidmolecule to form a duplex with the third nucleic acid molecule issubstantially different from the ability of the second nucleic acidmolecule to form a duplex with each other member of the family, whereinthe second nucleic acid molecule is suitable for use as a probe when theligand concentration at which the ability of the second nucleic acidmolecule to form a duplex with the third nucleic acid molecule issubstantially different from the ability of the second nucleic acidmolecule to form a duplex with each other member of the family and theligand concentration at which the ability of the second nucleic acidmolecule to form a duplex with each member of the family issubstantially the same as its ability to form a duplex with each othermember of the family are substantially equal to each other.
 8. Themethod of claim 7 wherein the second nucleic acid molecule is suitablefor use as a probe when the ligand concentration at which the ability ofthe second nucleic acid molecule to form a duplex with the third nucleicacid molecule is substantially less than the ability of the secondnucleic acid molecule to form a duplex with each other member of thefamily and the ligand concentration at which the ability of the secondnucleic acid molecule to form a duplex with each member of the family issubstantially the same as its ability to form a duplex with each othermember of the family are substantially equal to each other.
 9. Themethod of claim 7, further comprising the step of (e) repeating steps(c), (d), (i) and (ii) for a second ligand.
 10. The method of claim 9,further comprising the steps of (c), (d), (i) and (ii) in the presenceof both the first and second ligands.
 11. The method of claim 1 whereineach of the first nucleic acid molecules is selected from a groupconsisting of a genetic sequence of a first virus and variants thereofknown to exist in nature.
 12. The method of claim 7 wherein each of thenucleic acid sequences of the first nucleic acid molecules is selectedfrom a genetic sequence of a first virus and variants thereof, and thenucleic acid sequence of the third nucleic acid molecule is selectedfrom a group of genetic sequences known to exist in nature exclusive ofthe first virus and the variants.
 13. A method of detecting the presenceof a nucleic acid molecule suspected of being in a sample containinggenetic material, the method comprising: (a) providing the sample whichcontains or possibly contains a nucleic acid molecule which is a memberof a family of first nucleic acid molecules that is related to all othermembers of the family by a consensus sequence; (b) exposing the sampleto a probe comprising a nucleotide sequence having a sequencecomplementary to the consensus sequence and identified according to themethod of claim 1 as suitable for use in a probe, under conditionssuitable for hybridization, in the presence of the ligand of claim 1present at the concentration at which the ability of the second nucleicacid molecule to form a duplex with each member of the family issubstantially the same as its ability to form a duplex with each othermember of the family; and (c) ascertaining whether any duplexed nucleicacid molecules comprising the probe formed in step (C), wherein theformation of the duplexed nucleic acid molecules indicates the presenceof the nucleic acid molecule suspected of being in the sample.
 14. Themethod of claim 13, comprising the further step of exposing the same ofto conditions suitable for amplifying the duplex formed in step (B). 15.A method of promoting the hybridization of a nucleic acid capture moietyto a target single-stranded nucleic acid sequence and all its familymembers without hybridizing to a plurality of other non-target partiallycomplementary nucleic acid sequences present in a sample, the stepscomprising: (a) providing: (i) a nucleic acid capture moiety comprisingthe nucleic acid molecule identified in claim 5; (ii) the samplecontaining the target duplex nucleic acid sequence or any of its familymembers, wherein the sample has been treated such that all duplexnucleic acid sequences present in the sample including the target duplexnucleic acid sequence and all its family members suspected of beingpresent in the sample will denature and form single-stranded nucleicacid sequences; and (iii) a nucleic acid sequence binding ligand; (b)forming a reaction mixture comprising the nucleic acid capture moiety,the sample and binding ligand of steps (a)(i), (a)(ii) and (a)(iii),respectively under conditions such that the nucleic acid sequencebinding ligand promotes hybridization of the target single-strandednucleic acid sequence and all its family members to the nucleic acidcapture moiety comprising the probe nucleic acid sequence identified instep (a) and not to other non-target partially complementary nucleicacid sequences; (c) allowing the target single-stranded nucleic acidsequence and all its family members suspected of being present in asample to hybridize to the nucleic acid capture moiety comprising theprobe nucleic acid sequence identified in step (a) such that the nucleicacid sequence binding ligand promotes hybridization of the targetsingle-stranded nucleic acid sequence and all its family members to thenucleic acid capture moiety comprising the probe nucleic acid sequenceidentified in step (a) without promoting the hybridization of othernon-target partially complementary nucleic acid sequences; and (d)detecting the presence or absence of the target single-stranded nucleicacid sequence and all its family members hybridized to the nucleic acidcapture moiety.
 16. A method of promoting the hybridization of a nucleicacid capture moiety to a target single-stranded nucleic acid sequenceand all its family members without hybridizing to a plurality of othernon-target partially complementary nucleic acid sequences contained in asample, the steps comprising: (a) identifying at least one nucleic acidsequence probe substantially complementary to the target duplex nucleicacid sequence and all its family members suspected of being present in asample and coupling at least one probe nucleic acid sequence with alabel; (b) identifying at least two substantially complementary primernucleic acid sequences immediately 5′ and 3′ of the region of the targetnucleic acid sequence and all its family members to be detected; (c)treating the sample suspected of containing the target duplex nucleicacid sequences and all its family members with the two substantiallycomplementary primer nucleic acid sequences in step (b), an agent forpolymerization, and four nucleoside triphosphates under conditions whichallow amplification of the nucleic acid sequence to be detected thereofwithout simultaneous amplification of non-target partially complementarynucleic acid sequences from other viral nucleic acid sequences or humangenomic nucleic acid sequences; (d) treating the sample such that allduplex nucleic acid sequences present in the sample including theamplified target duplex nucleic acid sequence and all its family memberssuspected of being present in the sample denature and formsingle-stranded nucleic acid sequences; (e) providing: (i) a nucleicacid capture moiety comprising the labeled probe nucleic acid sequenceidentified in claim 5; (ii) the sample suspected of containing theamplified single-stranded target nucleic acid sequence and all itsfamily members; and (iii) a nucleic acid sequence binding ligand; (f)forming a reaction mixture comprising: (i) the nucleic acid capturemoiety comprising the labeled probe nucleic acid sequence identified inclaim 5; (ii) the sample suspected of containing the amplifiedsingle-stranded target nucleic acid sequence and all its family members;and (iii) a nucleic acid sequence binding ligand; under conditions suchthat the nucleic acid sequence binding ligand promotes hybridization ofthe amplified target single-stranded nucleic acid sequence and all itsfamily members to the nucleic acid capture moiety comprising the probenucleic acid sequence identified in step (a) and not to other non-targetpartially complementary nucleic acid sequences; (g) allowing theamplified target single-stranded nucleic acid sequence and all itsfamily members suspected of being present in a sample to hybridize tonucleic acid capture moiety comprising the probe nucleic acid sequenceidentified in step (a) such that the nucleic acid sequence bindingligand promotes hybridization of the target single-stranded nucleic acidsequence and all its family members to the nucleic acid capture moietycomprising the probe nucleic acid sequence identified in step (a)without promoting the hybridization of other non-target partiallycomplementary nucleic acid sequences; and (h) detecting the presence orabsence of the target single-stranded nucleic acid sequence and all itsfamily members hybridized to the nucleic acid capture moiety.
 17. Themethod of claim 16 wherein the nucleic acid sequence binding ligand isselected from the group consisting of: a compound which binds to aduplex nucleic acid in a sequence-specific way; a compound which bindsto a duplex nucleic acid in a non-specific way; a protein; an enzyme; anenzyme which alters the structure of a duplex nucleic acid to which itbinds; an enzyme which alters the structure of a duplex nucleic acid towhich it binds by breaking or forming a covalent or non-covalent bond,between an atom of the nucleic acid and another atom; an enzyme whichcleaves one or both strands of a duplex nucleic acid to which it binds;a restriction enzyme; a restriction endonuclease; an enzyme whichmethylates the duplex to which it binds; an enzyme which alkylates theduplex nucleic acid to which it binds; a nucleic acid ligase such as DNAligase, an enzyme which promotes or catalyzes the synthesis of nucleicacid; a nucleic acid polymerase; a nucleic acid polymerase whichrequires a double stranded primer; a DNA polymerase; DNA polymerase I;Taq polymerase; an RNA polymerase; an enzyme which alters the primary orsecondary structure of a duplex nucleic acid to which it binds; atopoisomerase; an enzyme which promotes or inhibits recombination; a DNAbinding agent; a mutagen; a compound which enhances the expression of agene under the control of the duplex bound by a ligand; a compound whichinterrelates into a duplex nucleic acid molecule; a compound which, whencontacted with a reaction mixture comprising a first single strandednucleic acid molecule and a second single stranded nucleic acid moleculewill increase the free energy of duplex formation at least n-fold,wherein n is 2, 5, 10, 50, 100, 500, 103, 104, 105, 106, a compoundwhich, when contacted with a reaction mixture will decrease the freeenergy of duplex formation by at least n-fold, wherein n is 2, 5, 10,50, 100, 500, 103, 104, 105,
 106. 18. The method of claim 17, whereinthe nucleic acid sequence binding ligand further comprises asingle-stranded nucleic acid binding ligand.
 19. The method of claim 17,wherein the nucleic acid sequence binding ligand further comprises aduplex nucleic acid sequence binding ligand.
 20. The method of claim 17,wherein the nucleic acid sequence binding ligand further comprises anonspecific nucleic acid binding ligand.
 21. The method of claim 17,wherein the duplex nucleic acid sequence binding ligand is selected fromthe group consisting of actinomycin D, distamycin A, diminazeneaceturate, bisbenzamide, and ethidium bromide.
 22. The method of claim16 wherein the target nucleic acid sequence and all its family membersto be detected is a region of a viral nucleic acid sequence and all itsfamily members.
 23. The method of claim 22, wherein the region of viralnucleic acid to be detected is a region of the ADS virus and all itsfamily members.
 24. The method of claim 16, wherein the target nucleicacid sequence and all its family members to be detected is a region ofan oncogene.
 25. The method of claim 24, wherein the oncogene isselected from the group consisting of p53, ras, BRCA1, or BRCA2 and eachof their family members.